Summary auto-generated
This study identifies the phosphorylation sites of human parainfluenza virus type 1 (HPIV-1) P protein, a component of the viral RNA polymerase. Using phosphopeptide mapping and site-directed mutagenesis in virus-infected and P gene-transfected cells, researchers found that HPIV-1 P protein is constitutively phosphorylated primarily at serine-120 and secondarily at serine-184. Importantly, phosphorylation at serine-120 depends on the presence of proline-121, forming a Ser-Pro motif similar to that found in the related Sendai virus P protein. Although HPIV-1 and Sendai virus P proteins share only 54% amino acid identity and have different phosphorylation site locations, both are primarily phosphorylated at serine residues followed by proline via proline-dependent protein kinases. This represents an unusual finding, as negative-sense RNA virus P proteins had not previously been shown to undergo phosphorylation by this kinase class. The conservation of constitutive phosphorylation at Ser-Pro sites across different parainfluenza virus isolates suggests functional importance for viral replication or polymerase activity.
Key findings
- HPIV-1 P protein is constitutively phosphorylated at Ser-120 (major site) and Ser-184 (minor site)
- Phosphorylation at Ser-120 requires proline-121, forming a Ser-Pro motif similar to Sendai virus despite low sequence identity between the viruses
- Both HPIV-1 and Sendai virus P proteins are phosphorylated by proline-dependent protein kinases, an unusual mechanism not previously documented for negative-sense RNA virus phosphoproteins
- The primary phosphorylation sites are conserved across multiple HPIV-1 clinical isolates
- P protein expressed in transfected cells is phosphorylated identically to that from virus-infected cells, indicating involvement of cellular kinases
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Abstract
RNA-dependent RNA polymerases of single-stranded, negative-sense RNA viruses comprise a phosphoprotein (P) and a large protein. The constitutive phosphorylation of the P protein in these viruses is highly conserved, yet the functional significance of phosphorylation is enigmatic. To approach this problem, phosphorylation sites were determined in two closely related paramyxovirus P proteins. Sendai virus (SV) is a prototypic paramyxovirus. Previously, using a phosphopeptide mapping technique, the primary constitutive phosphorylation site of SV P protein was mapped to Ser-249. Phosphorylation at Ser-249 is dependent on the presence of Pro-250. Human parainfluenza virus type 1 (HPIV-1) P protein has 66% similarity to SV P protein and its predicted secondary structure is highly similar to that of SV P protein. However, there is no obvious conserved phosphorylation site in HPIV-1 P protein. Using the phosphopeptide mapping strategy, the constitutive phosphorylation sites of HPIV-1 P protein were mapped. The HPIV-1 P protein is primarily phosphorylated at Ser-120. Phosphorylation at Ser-120 is dependent on the presence of Pro-121. It also has a minor phosphorylation site at Ser-184. The sequence at Ser-184 does not match any consensus phosphorylation target site for the known kinases. Significantly, the P proteins from both viruses are constitutively and primarily phosphorylated at one serine and the phosphorylation of that serine is dependent on the presence of a proline on its carboxyl side.